Import of peptides into intact cells is exceedingly difficult because of the low permeability of most peptides across biological cell membranes. This greatly restricts the use of synthetic peptides in regulating the biological functions of intracellular proteins and the development of therapeutic peptide drugs. The long-term goal of this study is to develop a peptide system by which intracellular processes such as signal transduction and gene transcription can be regulated by outside-in synthetic peptides. Such cell membrane-permeable peptides can be engineered by attaching a hydrophobic signal peptide sequence to a functional sequence. They can be translocated readily from the outside to the inside of the cells to influence cell functions. The specific objective of this proposal is to apply cell membrane- permeable peptides to regulate Ras activation by epidermal growth factor (EGF) or v-src oncogene. This Ras activation is mediated by an adaptor protein Grb2 containing both SH2 and SH3 domains. We focus on the regulation of the binding of Grb2 protein to its immediate substrates, a key link in the Ras activation cascade. To influence the cytosolic protein-protein interactions of Grb2 with its substrates including EGF receptor, Shc, and Sos proteins, cell permeable peptides containing short SH2 and SH3 binding sequences of these substrates will be designed and synthesized. Peptide import into NIH 3T3 and other cell lines will be determined by indirect immunofluorescence and radio-labeling assays. The inhibitory effects of these cell permeable peptides on Grb2 interaction with its immediate substrates will be examined by in vivo protein association assays. We will also determine the functional significance of these interactions in downstream Ras signaling by importing cell permeable peptides into cells to investigate their inhibitory effects on downstream Ras, Raf-1, and MAP kinase activations, cell proliferation and transformation. This study will present us with a novel, simple, and achievable approach for regulating intracellular processes in intact cells with synthetic peptides. It will also provide a better understanding of the Ras activation and signaling initiated by EGF and v-Src tyrosine kinase.